Microfluidic devices and methods for cell processing

ABSTRACT

Microfluidic devices and methods that use cells such as cancer cells, stem cells, blood cells for preprocessing, sorting for various biodiagnostics or therapeutical applications are described. Microfluidics electrical sensing such as measurement of field potential or current and phenomena such as immiscible fluidics, inertial fluidics are used as the basis for cell and molecular processing (e.g., characterizing, sorting, isolation, processing, amplification.) of different particles, chemical compositions or biospecies (e.g., different cells, cells containing different substances, different particles, different biochemical compositions, proteins, enzymes etc.). Specifically, the present invention discloses a number of sorting schemes for stem cells, whole blood and circulating tumor cells, and extracting serum from whole blood.

This is a divisional of non-provisional patent application entitled“Microfluidic Devices and Methods for Cell Sorting, Cell Culture andCells Based Diagnostics and Therapeutics,” Ser. No. 13/738,968 filed onJan. 10, 2013, which claims the benefit of and priority to provisionalpatent application Ser. No. 61/585,181 filed on Jan. 10, 2012. Thecontents of the above-mentioned applications are hereby incorporatedfully by reference into the present application.

STATEMENT OF GOVERNMENT INTEREST

This invention was made partly (iPS Cells sorting) with Governmentsupport under Contract No.W81XWH-12-C-0108 awarded by DoD and NIH. TheGovernment has certain rights in this invention.

BACKGROUND

The present application relates generally to medical devices and methodsand more particularly to microfluidic devices and methods for processingcells and molecules for therapeutics (stem cell therapy, gene therapy)and diagnostics (e.g., characterizing, amplification, sensing,processing, enriching circulating tumor cells.) using differentbiospecies (e.g., different cells, cells containing differentsubstances, different particles, different biochemical compositions,genes, proteins, enzymes etc.).

‘On-the-Fly Field-Potential Sensing Electrode Track’ Technology for StemCell Sorting

The derivation of patient-specific reprogrammed somatic cells makesimmunologically compatible stem cell replacement strategy veryattractive for several applications such as spinal cord therapy.Pluripotency has been derived at increased efficiencies from severaleasily accessible human cell types, including blood cells, keratinocytesand dermal fibroblasts enable drug screening, disease modeling andautologous cell transplantation in clinical therapy. The major challengein this tissue replacement therapy is the establishment of effectiveisolation of differentiated cells to avoid teratoma formation. However,at present, it is unclear whether any of the currently availablestrategies to generate differentiated cells from iPSCs is able toeliminate the risk of teratoma formation. Moreover, the currentseparation method—microscope-assisted manual isolation—is error-prone,time-consuming, and labor-intensive, and does have the capacity to sortmultiple cell phenotypes. Fluorescence-activated cell sorting is capableof sorting multiple phenotypes but uses labeling of cells.Magnetic-activated cell sorting also lacks the robustness to sort outmore than one cell type at a time. Unfortunately, conventionalseparation techniques requiring exogenous labeling or geneticmodification is not suitable for clinical applications and so superioriPSC sorting is an urgent unmet need. The novel flow based highthroughput label-free sorting scheme to separate stem cells and theirdifferentiated progeny for regenerative medicine using cell sortingbased on their response to electrical stimulation called ‘On-the-FlyField-potential Sensing Electrode Track’ (OFFSET) technology issignificant.

This high throughput integrated OFFSET platform can be configured to:

-   -   precisely flow and focus high throughput single-cells    -   accurately detect single cells using impedance sensing    -   stimulate the detected cells triggered by the impedance sensor    -   detect their stimulated response through an electrode array    -   process the stimulus response signature in real time    -   perform fluidic switching with the comparison of pre-stored        stimulus response signatures

In microfluidics, surface electrodes have been used to detect electricalsignals from cells such as extracellular ionic currents producing acharacteristic field potential (FP) signal in different ion channels andgating kinetics. Though these electrophysiological measurements havebeen used to identify subpopulations of electrically-excitable cells, atstatic conditions, high throughput flow based ‘on the fly’ recordinghave not done so far which are very essential for clinical applications.These FP signals can be monitored during stem cell differentiation andare characterized as a marker for endpoint analysis of embryonic stemcells in order to quantify ion channel expression levels and cellmaturity or phenotype. Our innovation is to develop a flow based systemto sense the degree to which stem cells have differentiated into thesecell types using ‘On-the-Fly Field-potential Sensing Electrode Track’(OFFSET) technology. Therefore OFFSET technology offers a label-freecell sorting of stem cells and their differentiated progeny based ontheir response to electrical stimulation with several advantages. Inpotential therapeutic applications, the cell populations relevant fortherapy can be electrically excited and the resulting transmembrane ioncurrents are measured using an array of surface microelectrodes alongthe direction of the flow. The electrical current measurements inresponse to electrical stimulation for differentiated states of thecells are built up as electrical signatures for real time comparison andsorting. Since these transmembrane ion currents are measurednon-invasively to sort the differentiated cells based on these fieldpotential markers, the sorted cells are highly viable for therapeuticapplications. Therefore ventricular-like cardiomyocytes from iPSCderived populations can be separated for cardiac tissue replacementtherapies in an ‘on the fly’ system at high throughput scale. These cellpopulations for such sorting include cardiomyocytes, neurons, skeletalmuscle, and vascular smooth muscle. The OFFSET platform combines thetechnologies of flow based field potential sensing, high speed signalprocessing and high throughput microfluidic cell sorting to rapidlydetect, identify, and sort millions of specific cells for downstreamapplications. Our endeavor to overcome the barriers that preventsuccessful translation of stem cell biology into clinical therapy ishighly significant to improve human health and control of humandiseases.

Flow Driven Blood Based Sorting of Cancer Cells Using Multi SpiralFluidic Channels

Sorting of cancer cells particularly circulating tumor cells (CTCs) fromblood is important for clinical diagnostics. Despite the progress inearly diagnosis and introduction of new therapy regimes, cancer remainsa prominent health concern in modern societies with one in four deathsin the US and a total of 1,529,560 new cancer cases with 569,490 deathsfrom cancer projected in 2010. The early dissemination of the cancer andthe systemic spread of tumor cells to other parts of the body results ina negative prognosis and death. Such CTCs can be found in the peripheralblood of patients before the primary tumor is detected. Therefore CTCsare fluid biopsy for primary tumor cells sampled as a minimallyinvasive, prognostic and predictive marker to reflect the biologicalcharacteristics of tumors and are implemented in an increasing number ofclinical studies. These CTCs play a pivotal role in changing the biologyand marker expression compared with the primary tumor and so detectionand characterization of these cells are believed to have a substantialclinical impact on the prognosis and optimal disease management ofcancer patients. In addition to a potential role in early diagnosis andprognosis, the detection of CTCs can guide therapeutic strategies forpersonalized treatment of patients with metastatic cancer.Identification, enumeration and characterization of CTC throughimmunocytochemistry, fluorescence in situ hybridization assays and allrelevant molecular techniques. However, the most challenging obstacle inthe separation and detection of CTCs is their extremely lowconcentration. Due to the rarity of the CTCs, existing immunomagneticcell separation techniques lack the ability to separate all types ofCTCs directly from whole blood at rapid and low cost.

Quantification of CTCs through the use of magnetic bead-conjugatedantibodies against epithelial-cell adhesion molecule (EpCAM) remains apoint of discussion for treatment decisions. EpCAM-dependent assays arebased upon the assumption that the presence of epithelial cells inperipheral blood indicates the presence of tumor cells. However,epithelial cells may be found in healthy donors and EpCAM-based assaysare not able to detect normal-like tumor cells. Moreover, certain tumortypes such as melanoma are not of epithelial origin which suggests thatEpCAM-based assays may be of limited use. Epithelial antigen may be loston CTCs due to the epithelial-mesenchymal transition (EMT), which isconsidered to be a crucial event in the metastatic process. Furthermore,CTCs must be isolated alive for testing their potential capacity toinitiate tumor formation in animal models and must become easilyaccessible to a large range of molecular biological analysis. Thereforeflow driven blood based inexpensive on-chip high performance sortingusing yoked channels (f-BIOPSY) system that can isolate, quantify, andanalyze circulating tumor cells from a blood sample under inertialfluidics conditions using successive approximation sorting method is ofgreat significance. The f-BIOPSY system as compared in Table 1 canprepare CTCs for analyzing relevant cellular and molecular biologicaltechniques for potential genetic abnormalities without using an antibodybased assay. The f-BIOPSY system could have a profound influence on theearly diagnosis, prognosis, early detection of relapse, and thedevelopment of new targeted strategies.

TABLE 1 Comparison of f-BIOPSY technology Veridex Xanapath BiopicoCharacteristics (Cell Search) (I-SCOPE) (f-BIOPSY) Throughput Low MediumHigh Specificity Low Low Medium Integration Low Low High Fluid handlingRequired Required Not required Sorting/Enrichment Immuno- Immuno-Inertial fluidics magnetic magnetic Multiplexity Low Low High Cellviability Low Low High

In the f-BIOPSY device, blood flow passes through a “yoked” spiralchannel using successive approximation method, allowing size-selectiveisolation of rare tumor cells under fully reproducible and standardizedinertial fluidics conditions. The f-BIOPSY device is a low-costinnovative technology with the aim of achieving isolation of tumor cellswithout the requirement for large and expensive apparatus and iscompared with other leading systems in Table 1. Compared previous spiralmethod, the clinically usable highly innovative f-BIOPSY system hasseveral advantages as characterized in Table 2. The cells isolated fromthe device are can be analyzed using all relevant cellular and molecularbiological techniques pertinent to the identification andcharacterization of CTCs and their potential genetic abnormalities. Thislabel free system can isolate living cells, allowing further tissueculture experiments. The goal of this cell sorting system is to avoidharming the cells during sorting so that high performance multianalytedetection using mRNA expression profiling can be achieved. Furthermore,tumor cells can be isolated without using an epithelial antibody-basedassay, suggesting that the f-BIOPSY device can be used for the isolationof a large spectrum of tumor cells, including cells of non-epithelialorigin. They can be used within clinical trials as a basis for earlytherapy stratification and monitoring to replace expensive and adverseradiological imaging techniques. The f-BIOPSY technology can besensitive and reliable to allow detection and analysis of CTCs routinelyfor the early diagnosis, risk stratification in the adjuvant setting,early detection of relapse, the development of new targeted strategiesand guiding treatment decisions.

TABLE 2 Innovation of f-BIOPSY System Specification Previous Methodf-BIOPSY System Spiral Single spiral Double spiral or multiple spiralsWidth Constant Decreasing on one spiral and increasing on other MainSimple channel Expansion structures in main channel channel Sample RBCand leukocytes Continuously RBC and other concentration are sorted outat cells are extracted out in the end the channel by-pass channels fromone spiral to another spiral channels Sorting Inefficient Efficient dueto increasing efficiency channel width Multianalyte Not accurateCascaded branched spiral sorting channels with defined geometry enablemultianalyte sorting Rapid sorter constant width makes Larger effectivewidth of slow sorting parallel spiral channels enables high flow ratesorting.

Serum Based Mobile Driven Analyzer for Rapid Tests

The advent of personalized diagnostic approaches demands highly flexibleanalytical devices to perform multiplexed analysis for determining awide range of disease biomarkers such as enzymes, antigens and nucleicacids and therapeutic agents. In this regard, lab-on-chip microfluidicdevices paved the way for miniaturized, self-standing analytical systemsand technological solutions for integration, multiplexing andprogramming of such biospecific reactions. Panel assays with “ad hoc”biomarkers for monitoring the health status and the drug efficacy in aspecific patient is an urgent need for these devices. With this respect,these devices could be operated by a patient, a nurse, a physician or atechnician directly in the doctor's office or at the patient's bed torapidly provide all the clinical chemistry information necessary foraccurate diagnosis and patient follow-up. With the increasing power ofcomputation, communication and versatility of smart phones, in recenttimes, it is useful to configure the smart phones with fluidic devicesfor point of care diagnostics. However, performing such rapid bloodbased diagnostics would require development of appropriate technologiesfor preprocessing blood and measurement for personalized assays.Therefore, Serum based Mobile driven Analyzer for Rapid Tests (SMART) isof great significance. This effort can separate serum from whole blood,to perform diagnostics and to interface with smart phone to tap itspower through cloud computing and making the diagnostics informationavailable in private networks for its better utilization. In thissystem, personal diagnostics information from whole blood is derivedusing a microfluidic chip and transmitted to cloud computing network foraccess to relevant users. The innovations are as follows:

-   -   1. The SMART system can utilize a rapid serum sorting scheme for        colorimetric diagnostics of test panels from serum.    -   2. Rapid pumping of serum in the SMART system is accomplished by        acoustic steaming driven integrated passive pump.    -   3. Colorimetric measurement imaging is accomplished by        integrated lensless optics so that compact smart phone camera        can be utilized for quantification.    -   4. Computation, analysis and communication of the diagnostics        data to a cloud computing system is enabled by the smart phone        for remote multiuser access.

The innovations in SMART system combines the technologies of serumseparation in spiral fluidic channel, acoustic cavitations streamingpump, colorimetric assay for test panels, smart phone based imaging andhealth information communication through cloud computing to develop apowerful platform for space exploration related point of carediagnostics and commercialization. The system could carry out in aquick, multiplex diagnostics in a cost-effective fashion for crew healthmonitoring and clinical diagnostic or therapeutic purposes. As anexample, simultaneous quantitative analysis of glucose, lactate, anduric acid in blood samples is applied.

The device allows short analysis time due to miniaturization and highflexibility of assay and format, as well as a potential costs reduction.Its key advantage is that the different types of assays described abovecan be performed simultaneously exploiting microarray configurations.This multiplexing capability can permit the development of chips for thedetection of panels of diagnostic biomarkers, e.g., for performing thediagnosis of an infectious disease by detecting both the pathogennucleic acids and the host antibodies produced in response to theinfection or to assess the liver function by combining routine clinicalchemistry analyses (bilirubin, cholesterol) with the measurement ofserum enzyme activities (alkaline phosphatase, aspartate, and alanineaminotransferase) and the detection of a present or past hepatitis viralinfection. Thanks to the flexibility of chip fabrication, chips toevaluate panels of biomarkers could be also specifically developed for agiven patient to perform “personalized medicine” on the basis of geneticapproaches.

Parallel Incubators with Loaded Single Cells for Lysis and AmplificationReactions

While the cell is recognized as a fundamental unit that can generate acomplex organism containing cells with diverse patterns of geneexpression, only a limited number of measurement techniques permitsingle cell resolution that is interesting to the biological, medical,and pharmaceutical communities. Traditional methods of gene expressionanalysis examine pooled mRNA from thousands of cells, resulting in anaveraged picture of gene expression across an entire cell population.This restricts the ability to distinguish between the individualresponses inherent cell-cell heterogeneity within a sample and todisentangle the complexity of the regulatory mechanisms controllingspecific responses. The primary problems that hinder such single cellanalyses are difficulty in handling a minute amount of sample, inabilityto prepare and manipulate single cells, inability to havehigh-throughput capability, and not being able to integrate withamplification protocols and detection mechanisms. Further, theapplication of current single cell PCR techniques is limited by longturnaround times, high cost, labor intensiveness, the need for specialtechnical skills, and/or the high risk of amplicon contamination. Theneed for single-cell mRNA analysis is evident given the vast cellularheterogeneity of all tissue cells and the recent developments in wholegenome amplification procedures, single-cell complementary DNA arraysand single-cell comparative genomic hybridization. When genetic analysisof single cells becomes a common practice, new possibilities fordiagnosis and research can open up. Current attempts to performhigh-throughput single cell dispensing and analysis involves flowcytometry and robotics. However, such systems are expensive as a routineresearch or diagnostic device. Therefore, Biopico Systems, Irvineidentified this opportunity to develop a ‘Parallel Incubators withLoaded single cells for Lysis and Amplification Reactions (PILLAR)’system to perform high-throughput quantitative single-cell geneexpression profiling. This system can provide unique informationcritical not only to the quality control and clinical translatability ofiPSCs for regenerative medicine but also to understand the relationshipbetween transcriptional and phenotypic variation in the development ofpathology, oncogenesis, and other processes of a target cell. Forexample, precise molecular analysis of abnormal gene and proteins insingle cells from a large population of cells helps in cell clonality,genetic anticipation, single-cell DNA polymorphisms and early diagnosisof cancer, infectious diseases and prenatal screening.

PILLAR technology, first traps single cells at configured micropillarsand then encapsulates using immiscible fluidics around the single celltraps as picoliter reservoirs. The novelties pertaining to thedevelopment of the PILLAR system include 1) trapping of single cellswith a set of micropillars (and guiding pillars for focusing the cellstowards traps) 2) formation of microreactors using immiscible fluidsaround a single cell and 3) thermocyling of these anchored microreactorsusing flow of oil at two different temperatures for fast and reliablePCR reaction. The all-in-one PILLAR system combines commerciallyavailable fluorescent RT-PCR with immiscible microfluidics and singlecell microtrapping technologies for performing thousands of single cellRT PCR in picoliter volumes in a quick, high-throughput, andcost-effective fashion. The PILLAR system can help to understand therelationship between stochastic variations of gene expression withinindividual cells and heterogeneous transcriptional profiles across apopulation of cells.

This highly integrated PILLAR platform is configured:

-   -   to precisely trap high throughput single-cells in array of        micropillar based trapping sites    -   to encapsulate single cells as picoliter reactors by flow of        immiscible fluids    -   to rapid thermal cycling on anchored single droplets at the        micropillar trapping sites    -   to perform thousands of single-cell PCR for regenerative        medicine or clinical diagnostics        This PILLAR platform would be very useful for accurately        quantifying the differentiation process and could serve as a        performance metric of every step of stem cell differentiation        process for regenerative medicine. The combination of        high-fidelity manipulation of single cells and the ability to        perform nucleic acid amplification offers the possibility of        developing a powerful automated instrument which has highly        significant commercial applications such as cancer diagnostics        and prenatal diagnostics.

Biochemical analysis of genes and proteins from single cells is ofsignificant interest. The intellectual merit of the technology is todevelop a chip/system that entraps single cells and performs 10,000parallel single cell RT-PCRs simultaneously in a cost-effective fashion.This can provide a means to perform precise molecular analyses on singlecells from large populations of cells using a highly sensitive approach.The chip combines three unique microfluidic techniques for theautomation of a cell-based real-time PCR-based diagnostic system on achip with supporting analytical instrumentation. This novel device candifferentiate gene expression of a particular rare cell from othersingle cells with a capability of multiplexing for the detection ofhouse-keeping genes and target genes. This capacity to analyze largepopulations of individual cells could provide unique opportunities inthe life sciences and support biomedical research activities in thefields of virology, oncology and pathology.

The broader/commercial impact of the project is the development of acost-effective solution for diagnosis of various diseases using cellulargene expression variation of multiple single cells for leukemia,prenatal screening, genetic screening of multiple diseases, or thedetection of viruses such as HIV virus. Isolation of single cell in ahigh throughput fashion followed by gene expression analysis can lead toseveral research findings. Such findings can lead to early detection andprognosis of diseased states including differential detection of aninfected cell from uninfected cells, detection of cancer markers indifferent cells, and changes in gene expression of diseased single cellsat high speed and high specificity. Further, small concentration changesand/or altered modification patterns of disease-relevant components,such as mRNA and/or micro RNA, have the potential to serve asindications of the onset, stage, and response to therapy of severaldiseases. In current, manual, single-cell PCR methods low abundance mRNAis often lost during cell lysis and extraction processes and thesemethods are also extremely labor intensive requiring expensive equipmentto isolate single-cells to perform PCR. The PILLAR system can detectrare abnormal cells and carry out single-cell PCR or RT-PCR. Micrototalanalysis systems of the prior art have typically involves microdropletsformation followed by cells and molecules encapsulation. Thesetechnologies require external active instruments to accomplish medicaldiagnostics which can be difficult to perform one touch or one stepdevice operation. On the other hand, in our technology, single ormultiple cells or molecules are trapped first either at configuredmicropillars or nozzles and then picoliter volume reservoirs or reactorsare formed around the single cell using immiscible fluidics. Theisolated single cell or cells or molecules are processed for multisteptemperature reactions or chemical reaction. Multiple temperaturebiochemical reaction such as PCR or linear isothermal amplification canbe carried out on the trapped picoreactors by flowing immiscible fluidssuch as oil around the picoreactors. Further, if needed, a slip and lockchip technique supplies additional reagent from another layer ofmicrofluidic chip. PCR using fluid flow thermal cycling is also highlyinnovative. The dimensions of the pillars for trapping cells and flowrate of the oil for encapsulating single cells are optimized for theefficiency and specificity of the diagnostics device.

Programmable Array of Living Cells for Combinatorial Drug Screening

Combinatorial drug screening for cell based drug discovery and efficacyis increasingly dependent on high-throughput technologies due to theneed for more efficient screening of multiple combinatorial drugcandidates. Massively parallel analytical screening technologies areneeded for the exploitation of biological insight in the oncology clinicsince cellular responses to anti-cancer modalities have been stochasticin nature. Miniaturized reactors have been developed to reduce culturevolume, increase process efficiency and to administer chemotherapeuticdrugs sequentially or together in combination for massive experimentalparallelization of real-time drug screening routines. The use ofcombination therapies can lead to increased efficacies at significantlylower doses and side-effects and so investigation of combinationtherapies for curative and palliative care is very significant.Therefore an automatic Programmable Array of Living cells (PAL) thatintegrates on-chip generation of drug concentrations and pair-wisecombinations with parallel culture of cells is significant for drugcandidate screening applications.

SUMMARY

In accordance with the present invention, there are provided methods forstem cell sorting using flow based field potential sensing and sorting,circulating tumor cell sorting using multiple spiral channels, cellculture using on-chip pump and cell based assays in microfluidics, bloodserum separation for smart phone based diagnostics, multiplexed singlecell PCR using multiple pillars and immiscible fluidics

In accordance with the stem cell sorting invention three sets ofelectrodes are employed for impedance sensing, stimulus current anddiscrete recording of time domain stimulus response using an array ofelectrodes (20 pairs or more) in the path of flow cells in order todistinguish between undifferentiated cells and different differentiatedspecies. Still further in accordance with the present invention, thereare provided methods for the extraction of sheath fluid flow fromdilution of the sorted cells and deflecting fluidic inlets for highspeed sorting using high pressure fluidic pulse.

In accordance with the circulating tumor cells sorting invention, thereare provided methods employing a spiral microfluidic channel systemhaving one or more spiral microfluidic channels with periodicallyinterconnected channels called “yoked channels”. Cells are delivered atan inlet, then flowed through a primary spiral microfluidic channel andone or more secondary spiral microfluidic channels, and collected at oneor more outlets. For example, CTCs can be separated from a blood samplehaving, among other cells, erythrocytes, leukocytes and CTCs using thespiral microfluidic channel system.

Still further in accordance with the present invention, there areprovided methods with one or more spiral microfluidic channels withwidth gradients. Such method can have decreasing width gradient periodicpinching regions to sort cells at high efficiency. While one spiralchannel can have decreasing width gradient, another may have increasingwidth gradient in order to provide balanced pressure profile. Forexample, a primary spiral microfluidic channel has a greater width atits inlet than at its outlet, while a secondary spiral microfluidicchannel has a smaller width at its inlet than at its outlet.

Still further in accordance with the present invention, there areprovided methods with connecting channels (yoked channels orinterconnect channels) between two successive and concentric spiralmicrofluidic channels whose widths depend on the size of the cells orparticles or species under sorting.

Still further in accordance with the present invention, there areprovided methods with periodic pinching regions whose length depends onthe flow rate of the fluidics. The lengths of the pinching regions arealso increasing from the center of the spiral microfluidic channelsystem towards outlets of the spiral microfluidic channels.

Still further in accordance with the present invention, there areprovided methods for off-chip pre-processing and on-chip processing ofcells.

Still further in accordance with the present invention, there areprovided methods using multiple species sorting with multistep forseparating different species of blood and for isolating CTCs ofoverlapping sizes with the peripheral blood leukocyte size scale.

In accordance with the serum based smart phone driven diagnosticsinvention, there are provided methods for separating serum from wholeblood using dual spiral channels. In one channel serum is separated dueto geometric singularity at the dead zone and is transferred to theadjacent spiral channel through connecting channels.

Still further in accordance with the present invention, there areprovided methods for clinical diagnostics using colorimetric assay orfluorescent assay or other spectroscopy to perform serum based multistepchemistry on microarray spots in cellulose pad, image microarray spotsusing extended optics on smart phones, quantify the assay using imageprocessing and transmit the results to cloud computing server forhealthcare providers or other personals.

Still further in accordance with the present invention, there areprovided methods for integrating software for optics, imaging andmechanical hardware, software for communicating to cloud portal,software for image processing, software for phone application andinteraction with users.

Still further in accordance with the present invention there are methodsprovided for extraction of serum from blood using multiple spiralchannels

In accordance with the single cell PCR invention, there are providedmethods for encapsulation of cells or molecules in trapping sites suchas configured micropillars or micronozzles using immiscible fluids suchas oil, fluorinert, oil containing surfactants, gel or other medium.

Still further in accordance with the present invention, there areprovided methods for multistep thermal cycling for PCR or otheramplification by flowing of immiscible fluids as mentioned about atdifferent temperature.

Still further in accordance with the present invention, there areprovided methods for performing multistep chemical reaction usingmodified slipchip.

Still further in accordance with the present invention, there areprovided methods for fabrication of the chips with microarray driedspots of primers or any other reagent or immobilized on semi-sphericalgel on one layer and trapping of single cells with 6 sets of configuredmicropillars in another layer

Still further in accordance with the present invention, there areprovided methods for the assay system for diagnostics in frequencydomain by performing thousands of biochemical reactions and counting thepositives for the quantification.

Still further in accordance with the present invention, there areprovided methods for performing additional movement of trappedpicoreactor droplet using electrowetting on dielectric.

Still further in accordance with the present invention, there areprovided methods for carrying out electrophoresis in the medium of gelbased immiscible fluids.

Still further in accordance with the present invention, there areprovided methods for preprocessing samples such as blood, tissue, tumorsetc using cascaded magnetodiffusion or compounded flow focusing spiralinertial microfluidic based cell sorting.

Still further in accordance with the present invention, there areprovided methods for the formation of single cell encapsulated dropletsor various molecules encapsulated droplets undocked from the trappingsites for further processing.

Still further in accordance with the present invention, there areprovided methods for separation or purification of constituents of thedroplets such as mRNA or other species using magnetic beads throughmultistep processing of droplets such as cascaded fusion and fissionsteps.

Still further in accordance with the present invention, there areprovided microfluidic devices for carrying out the above-summarizedmethods.

A microfluidic device according one implementation of the presentinvention generally comprises a) at least six set of micropillars in aconfiguration to trap cells electrodes positioned parallel to thedirection of flow, b) apparatus (e.g., on chip pump or micropumps) forapplying a flow for oil and aqueous fluids and c) apparatus formeasuring the biochemical reaction (e.g., fluorescent microcope,integrated fluorescence reader, other optical reader, GMR sensor,impedance sensor, nanosensor). (d) pair of electrodes to carry outelectrophoresis in gel after amplification of molecules or genes (e)This device may comprise a microfluidic device that has a substratelayer and an upper layer, wherein the electrodes are located (e.g.,fabricated, formed, affixed to or otherwise disposed on or in) one ofthe layers (e.g., on the substrate layer) and the microchannel islocated (e.g., fabricated, formed, affixed to or otherwise disposed onor in) in the other layer (e.g., in the upper layer). The layers of thedevice may be fully or partially formed of different materials. Forexample, the layer in or on which the electrodes are located (e.g., thesubstrate layer) may comprise a glass and the layer on or in which themicrochannel is located (e.g., the upper layer) may comprise a suitablepolymeric material such as polydimethylsiloxane (PDMS), polycarbonate,polyacrylate, COC etc.

Still further in accordance with the present invention, there areprovided methods for transporting samples and reagents usingelectrowetting on dielectric actuation in nanodroplets.

Still further in accordance with the present invention there are methodsfor encapsulating single or multiple cells along with nutrients andmedia and label molecules in different reactors and monitoring theirgrowth using fluorescence or electrical impedance or any other methods.

Further aspects, elements and details of the present invention aredescribed in the detailed description and examples set forth here below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1B Schematic configuration of the stem cell sorting fluidic andelectrodes array system. Representation of electrode array for impedancesensing, stimulus current and discrete recording of time domain stimulusresponse using 20 electrodes in the path of flow cells.

FIG. 2A-2C Schematics of electrodes, channel and OFFSET chip.

FIG. 3 Schematics of OFFSET Manifold for fluidics and electricalinterface.

FIG. 4A-4C Electronics Integration. Planar electrodes in the channel,wirebonded multichannel bare die amplifier, electrodes and fluidicspackage for signal cell detection.

FIG. 5 Field potential based characteristics of undifferentiated anddifferent differentiated species of iPSC.

FIG. 6 Flow chart showing the operation of the stem cell sorting system

FIG. 7 Complete schematics of OFFSET system.

FIG. 8 Prototype OFFSET System.

FIG. 9 Geometrical schematics of CTC sorting and diagnostics (f-BIOPSY)chip with width gradient, periodic pinching regions in spiralmicrofluidic channels.

FIG. 10 CTC sorting and diagnostics System for cancer diagnostics.

FIG. 11A-11B Optimization parameters of f-BIOPSY Chip. 3-D geometry ofthe portion of the f-BIOPSY chip.

FIG. 12A-12B CTC enrichment using decreasing width gradient periodicpinching regions in spiral channel inertial fluidics. Modified f-BIOPSYchip to isolate all CTCs of overlapping sizes using multistep sorting ofblood.

FIG. 13 Digital Centrifuge: Multiple size particulate sorting with 5spirals.

FIG. 14A-14E Schematic diagram of a serum based mobile driven rapid test(SMART) system. The system takes blood sample at the inlet of SMART chipfrom finger tip and docks at the cell phone. Serum from blood isseparated using a spiral channel actuated by acoustic cavitationsstreaming pump. Serum reacts with the reagents for colorimetric assay.Imaging is carried out using a transmission mode lensless optical setuplit by an LED. Smart phone performs computation of health data andtransmit to a cloud computing server.

FIG. 15A-15B Schematic diagram of a SMART chip for serum separation fromwhole blood and colorimetric assay. Serum separation spiral microfluidicchannel is actuated by acoustic cavitations streaming pump.

FIG. 16 Schematics of serum separation from whole blood using doublespiral microfluidic channel.

FIG. 17 Schematics of serum separation from whole blood using filters inspiral microfluidic channel.

FIG. 18 Schematic diagram of a SMART system hardware for personaldiagnostics.

FIG. 19 Schematics of a smart phone for software control, communication,computation and display.

FIG. 20A-20E Schematic diagram of a PILLAR chip/system that can trap andencapsulate thousands of single cells in configured micropillars andperform RT PCR. The operational steps are trap single cells, supply PCRmix and lysis buffer to trapped cells, encapsulate of single cells usingimmiscible fluidics, thermal cycle by flowing oil from hot/cold bathswith synchronized imaging for gene expression profiling and Dataprocessing and Statistical analysis for regenerative medicine orclinical diagnostics.

FIG. 21 Aqueous flow showing cells trapped at the micropillar trappingsites in a compartment of the parallel incubators single PCR (PILLAR)chip.

FIG. 22 Fluidics diagram of the massively parallel picoreactor chip withcells and reagents are flowed from the top inlet. Extra cells or cellclusters adhered weakly near the trap sites are cleaned by the flow atthe side inlet.

FIG. 23A-23E Successive frames of the formation of encapsulatedreactors. Successive frames of thermal convection profile from atemperature of 27° C. to 92° C. in 20 ms. Velocity of flow profileinside the chip with the application of a flow rate with a speed of 0.1m/s. Kinetics of temperature profile during the convective diffusion ofoil from a temperature of 27° C. to 92° C. (for PCR thermal cycling).

FIG. 24A-24B single cells trapping at a flow of cells at 1 uL/min,encapsulation of aqueous fluid using immiscible fluid at a flow of 5uL/min.

FIG. 25 Aqueous flow showing cells trapped at the micropillar trappingsites in a compartment of the PILLAR chip.

FIG. 26 Fluidic manifold for holding the PILLAR chip and flow basedthermal cycling using a set of valves and pumps.

FIG. 27A-27E The picoreactor system in slip and lock platform. Theoperation steps are (i) Flow of cells for trapping and valving of singlecells (ii) flow of oil to encapsulate the single cell in a picolitervolume compartment (iii) Flow of cell lysis/PCR master mix solutions(iv) Slipped chip to flow the add the reagents for PCR reaction. B. Theoperation steps are Cell encapsulation with oil flow, Thermal cycling:flow of hot oil, Thermal cycling: flow of cold oil, Real time PCR withfluorescent imaging.

FIG. 28A-28D Results of the flow analysis in a compartment in cellmicronozzle cell trap chip using CFD simulations. Velocity profile ofthe fluid flow. After the encapsulation of single cells with oil.Pressure developed inside the channel due to immiscible flow. CFDresults showing single cell trapping and flow blockage for successivecells.

FIG. 29A-29B Schematics of mechanical system for modified slip chipoperation using ‘slip and lock’ stage movement during fluidic delivery.Fluidics operation of slip and lock Chip.

FIG. 30 Digital output for bcr-abl rearrangement detection and positivecontrol (GAPDH).

FIG. 31A-31C Single cell diagnostics. Representation of encapsulation ofa single cell trapped by a configured array of 6 micropillars.Picoreactor droplet is coupled with EWOD to move the single cellencapsulated droplet for further serial processing. In-situelectrophoresis after PCR amplification in gel medium using a pair ofelectrodes fabrication on the bottom glass substrate.

FIG. 32A-32L Schematic diagram of a programmable array of living cellssystem. Full Chip Diagram. Fluidic operations of the chip areillustrated in the in the in-pictures. Flow of cells through the chip totrap cells at the ‘cup’ shaped reactor till it overflows. Flow of bufferto clean up the cells in the channels and incubate for 24 hours. Flow ofcombinatorial drug as a 2-D. Flow of oil to form cells encapsulatedreactors in isolation. Cell culture for 24 hours with Florescenceimaging of cell growth monitoring every 6 hours. Time lapse frames ofaqueous cells in compartments isolation by virtual walls are shown.Alternate diagram featuring complete isolation of cells from immisciblefluidics is shown.

DETAILED DESCRIPTION AND EXAMPLES ‘On-the-Fly Field-Potential SensingElectrode Track’ Technology for Stem Cell Sorting

The OFFSET disposable chip (shown in FIG. 1A) utilizes sheath flowfluidics, single cell impedance sensing, on the flow stimulation andrecording of response from individual cells and flow switchingmicrofluidics for rapid sorting thousands of iPSC. The chip includes (a)sheath flow inlets, (b) impedance sensor electrodes, (c) stimuluscurrent electrodes, (d) array of stimulus response recording electrodes(e) fluidic switching outlets. In the OFFSET chip has channel of width50 um and height 20 um with three inlets and 5 outlets and a 24 pairs ofPt or Au electrodes on glass substrate with a distance of 50 um betweenthe electrodes. The first pair of electrodes for impedance measurementis 50 um wide while the stimulation electrode is fabricated to be 500 umso that each cell can stay at the stimulated field for 5 ms. The flowrate of the cells flow can be considered to be at a velocity of 0.1 m/s.FIG. 1B shows a representation of electrode array for impedance sensing,stimulus current and discrete recording of time domain stimulus responseusing 20 electrodes in the path of flow cells.

The OFFSET system includes a sheath flow fluidics, single cell impedancedetection unit, stimulus current circuit, current response recordingcircuit, cell sorting fluidics and software control. The sheath flowcontrol is facilitated with syringe pumps and cell sorting fluidics iscontrolled by a set of valves. The iPS cells are diluted and deliveredin to the core flow inlet. The detection of single cells by thedifferential impedance circuit triggers the stimulus current that lastfor a few millisecond in the channel electrodes. In the meantime thestimulus response field potential of the flowing cells are recorded bythe array of electrodes (described in FIGS. 2A-C) and the fieldpotential signature of the cell is reconstructed from the recordedelectrodes. A high impedance low noise multichannel differentialamplifier with artifact suppression algorithm for recording the signalsis employed. The field potential signatures are compared with theprerecorded response curves from the database and the cell is deflectedin to the corresponding outlet channel by the activation of the flowswitching valves. Several cell lines such as Fibroblast based iPS cells,cardiac cells, muscle cells is used at a concentration of 2000 cells peruL for sorting. In validation experiments, the electrical recording iscompared with parallel fluorescent based Calcein assay detectionexperiments. The real time fluorescence detection system includes aTungsten-halogen lamp as an excitation source and a CCD detector. FIG. 3shows the schematic of OFFSET manifold for fluidics and electricalinterface. OFFSET chip, electrical interface cards, pumps, valves,reservoirs for pressurized air and sorting fluid, pressure sensor andfluid refilling port. Electronics Integration is achieved using planarelectrodes wirebonded to a multichannel bare die amplifier along withthe fluidics package as shown in FIG. 4A-C.

The OFFSET system is characterized for the sorting efficiency, thepurity of the enriched sample, and the differentiated cell throughput.The pluripotency of sorted iPSCs from the OFFSET system is assessedusing standard protocol such as gene expression markers, antigenmarkers, and epigenetic markers. The performance metrics of theintegrated OFFSET system such as specificity, purity can be establishedby cell morphology, quantitative immunochemical and RT PCR assaymethods. Validation and verification of electrical signature basedsorting is carried out using parallel Calcein fluorescent dye signalrecording. The extracellular field potential experiments for differentspecies of neural stem cells, cardiac cells, neurons, glia, muscle cellsunder static flow conditions is performed in order to compare with theflow based system. Cell sorting is performed after distinguishingbetween undifferentiated and differentiated stem cells and to achieve athroughput of 100 cells per second using much diluted sample. Theintegrity of targeted cells is evaluated by comparing the morphology ofcells before and after sorting using OFFSET system. The cell productsterility is assessed using standard clinical laboratory techniques.Commercially viable scaled up prototype can be built at 100% specificitywith a throughput of 1000 cells per sec for sorting different celllineages for different clinical iPS cell based therapies. An example offield potential signal for undifferentiated stem cells anddifferentiated cells is shown in FIG. 5. The reconstructed fieldpotential signal is post-processed by an artifact suppression algorithmto eliminate any artifacts through a combination of templatesubtraction, linear filtering, and least squares exponential curvefitting. Based on automated analysis of field potential signature, theoutlet flow is switched to one of several output reservoirs usingexternal electromechanical valves or additional pressure flow fluidics.FIG. 6 depicts the operation of the OFFSET device and FIG. 7 shows thesystem integration.

TABLE 3 Cell sorting throughput of OFFSET system Length of sortingfluidics channel 500 mm Volume of fluid in the channel along with 1 2.5nL cell for sorting (50 mm × 100 mm × 0.5 mm) Number of cells in 1 mL400 Velocity of the cells flow 0.1 m/s Maximum flow rate of cells inlet0.5 uL/sec (50 mm × 100 mm) Number of cells sorted per second 200Average number of differentiated cells sorted 100 per second (assuming50% contamination) Average numbers of differentiated cells 1000 targeted to sort per second

The parameters such as the sorting efficiency, the purity of theenriched sample, and the differentiated cell throughput are evaluated.The efficiency of the sorting routine measures the ratio of successfullysorted target cells to the total number of target cells detected. Theefficiency depends on the sorting cell concentration, the nature of thecells, the flow rate of fluidics, and the electrical signal recordingand an efficiency of 95% is expected. The purity, or final cellfraction, of the enriched sample is also dependent on the signal tonoise ratio of the electrical FP signatures, rate of cell arrival,switching time duration, and the heterogeneity in the differentiation ofthe iPSC. It is determined that the optimal lengths of switching for thechips are determined to be 0.5 mm according to the sorting efficiencyand sample purity at various sorting durations. The cell sortingthroughput is an important aspect of the sorter for potentialapplications. Conventional FACS systems can achieve rates of 100 kcells/s while microfluidic cell sorters using fluidic switchingmechanisms are generally on the order of 10-100 cells/s. The cellsorting throughput of OFFSET system using the flow and cell parametersare calculated to be 100 cells/sec is presented in Table 3. As differenttherapeutic applications of stem cell therapy would require million tobillion number of iPS cells the system can be optimized to a throughputof 1000 cells/sec. FIG. 8 shows the prototype of an OFFSET chip.

Flow Driven Blood Based Inexpensive on-Chip High Performance SortingUsing Yoked Channels

The CTC sorting and diagnostics (f-BIOPSY) chip includes a spiralmicrofluidic system having at least two spiral microfluidic channels, anouter spiral microfluidic channel with decreasing width where bloodsample is introduced for sorting and an inner spiral microfluidicchannel with increasing width where RBC and leukocytes can be extracted.The spiral microfluidic channels are connected with “yoke” channelswhich allow only smaller particles to move from the outer spiralmicrofluidic channel to the inner spiral microfluidic channel due tousing dean flows and differential migration under inertial microfluidics(FIG. 9). The spiral microfluidic system also includes periodic pinchingregions of decreasing width (100-20 μm) in high aspect ratio 9-loopspiral microfluidic channel with a height of 100 μm to sort erythrocytesfrom leukocytes and to sort CTC from leukocytes in a convenient singlestep.

The f-BIOPSY system includes a preprocessing step for blood, a sortingstep in f-BIOPSY chip and CTC processing for cancer diagnostics. Thef-BIOPSY system as shown in FIG. 10 takes whole blood diluted to 1%-10%hematocrit sample to efficiently sort CTC. The sorted CTC cells areobserved at the collection chamber using a microscope and imaged usingimmuno-fluorescence assays. The CTCs are collected and processing formultianalyte protein expression profiling. The f-BIOPSY system can beintegrated with flow devices to power the inertial fluidics for sorting,microscopic imaging, PCR thermal cycler, and control & analysis softwarefor cancer diagnostics.

The characterization and evaluation processes involve f-BIOPSY chipperformance and f-BIOPSY system performance. The high-throughputf-BIOPSY chip can be evaluated for each step of processes for theoptimization of geometry parameters and characterizing the optimizeddevice for performance metrics by analyzing the specificity, accuracy ofthe sorted cells. CTC recovery rate and the efficiency of the devicewith hematocrit percentage are measured. Our goal is to achieve greaterthan 95% specificity and accuracy. This success can open uppossibilities for faster-than-ever, low cost, high-throughput moreefficient and more sensitive devices for multi-analyte improved cancerearly detection, diagnosis, prognosis and treatment monitoring.

As illustrated in FIG. 9, a spiral microfluidic channel system includesprimary spiral microfluidic channel 901 coupled to inlet 903, andsecondary spiral microfluidic channel 902 coupled to primary spiralmicrofluidic channel 901. As illustrated in FIG. 9, primary spiralmicrofluidic channel 901 includes pinching regions 911, where secondaryspiral microfluidic channel 902 is connected to pinching regions 911 ofprimary spiral microfluidic channel 901 through interconnect channels910. Primary spiral microfluidic channel 901 and secondary spiralmicrofluidic channel 902 are substantially concentric. As illustrated inFIG. 9, primary spiral microfluidic channel 901 has a decreasing widthfrom inlet 903 to outlet 905, while secondary spiral microfluidicchannel 902 has an increasing width from channel inlet 909 to outlet904.

As illustrated in FIG. 9, the spiral microfluidic channel system isconfigured to separate cells, such as CTCs 906, leukocytes 907 anderythrocytes (RBCs) 908, for example. Cells, for example, in a bloodsample are delivered at inlet 903, and flowed through primary spiralmicrofluidic channel 901 and secondary spiral microfluidic channel 902,and collected at outlet 905 of primary spiral microfluidic channel 901and outlet 904 of secondary spiral microfluidic channel 902. Asillustrated in FIG. 9, CTCs 906 are separated from other cells such asleukocytes 907 and erythrocytes (RBCs) 908 through primary spiralmicrofluidic channel 901 and collected at outlet 905, while leukocytes907 and erythrocytes (RBCs) 908 are collected at outlet 904 of secondaryspiral microfluidic channel 902.

The width of each interconnect channels 910 between two successivespiral microfluidic channels depends upon the size of the cells orparticles or species under sorting. The length of each periodic pinchingregions 911 depends on the flow rate of the microfluidics. The lengthsof the pinching regions can increase from the centermost pinch region912 to the outermost pinch region 913. In one implementation, pinchingregions 911 can be periodically disposed along a concave sidewall ofprimary spiral microfluidic channel 901. In another implementation,pinching regions 911 can be periodically disposed along a convexsidewall of primary spiral microfluidic channel 901.

FIG. 11A-B shows a 3-dimensional structure of a portion of a chip havinga spiral microfluidic channel system. The primary spiral microfluidicchannel 1101, secondary spiral microfluidic channel 1102, interconnectchannels 1103 and pinching regions 1104 are shown. The initial geometrycan comprise a 9-loop (100 μm to 20 μm)×100 μm (W×H) double spiralchannel with two separate outlets. The number of turns and the widthgradient are optimized for the high specificity of sorting. Thedecreasing width gradient periodic pinching regions can provide highefficiency. In order to achieve high specificity in the sorting of CTCsfrom whole blood the spiral widths (wii 1111, woi 1113, wif 1116, wof1117—widths of inner and outer spiral at the initial and finalpositions) are adjusted. Further the length of the expanded and pinchedchannels (lec 1114, lpc 1115) are also adjusted to increase theefficiency of the sorting of CTCs. The ‘pinching’ width is a key featureof the f-BIOPSY chip for the successful isolation of CTCs from blood.The contraction width along this pinching region is fabricated to becomparable (smaller) to the CTC diameter, ensuring that the cells areeffectively ‘squeezed’ as they traverse through the contractionchannels. The number of turns of the spiral channels (ssc 1118—size ofspiral channels) is also optimized for the high efficiency andspecificity of the sorted cells. The widths of side channel (wsc 1112)are adjusted to avoid any leakage of CTC in to the RBC waste channel. Tostudy the effect of aspect ratio, microchannels of height 75 mm, 100 umand 150 um can be fabricated yielding aspect ratios of 3.75, 5 and 7.5,respectively. The high aspect ratio microchannels preferentiallyequilibrate cells along the longer channel dimension and process thesample at higher flow rate, thereby increasing the throughput.

The operation of a f-BIOPSY chip is based on the phenomenon of inertialmicrofluidics in spiral microfluidic channel 1201 having pinchingregions 1202 in convex configuration 1202, where pinching regions 1202are disposed on a convex sidewall of spiral microfluidic channel 1201.As illustrated in FIG. 12A, a blood sample having CTCs, leukocytes andRBCs is delivered at inlet 1203, and separated at outlets 1204, 1205,1206, respectively. In f-BIOPSY microchannels, under the Poiseuille flowcondition, particles of varying sizes equilibrate at distinct positionsalong the microchannel cross-section under the influence of inertiallift and Dean drag forces. The high aspect ratio microchannel usingshear modulated inertial lift forces that efficiently equilibrates allthe cells along the channel side walls. The CTCs can be isolated fromwhole blood in periodic pinching regions of outer spiral channels. Asthe cells flow in the spiral channel the bigger or heavier cells undergohigher inertia and do not undergo deviation in their paths where assmall cells undergo deviation towards the expanding region. Theerythrocytes from blood can move towards expansion and are extracted into the inner spiral channel. The larger CTCs (RBCs ˜5 μm; leukocytes˜8-14 μm; CTCs ˜16-20 μm diameter) are collected at the outer outletwith 90-100% recovery. Due to size proximity of leukocytes, CTCscollected may contaminate with leukocytes. The decreasing width of theouter spiral channel helps in the improved specificity of the sorting ofCTCs. As illustrated in FIG. 10, a f-BIOPSY system may include f-BIOPSYchip 1001 for on-chip processing of cells and off chip 1002 for off-chippre-processing of the cells for cancer diagnosis from whole blood.

In order to operate the f-BIOPSY System, whole blood can be filtered forany clusters and diluted to adjust the hematocrit before introducinginto the f-BIOPSY chip. The sorted CTCs can be collected formultianalyte gene expression profiling based cancer diagnosis.

As with any size-based CTC separation technique, a major limitation ofthis device is its inability to isolate CTCs that overlap the peripheralblood leukocyte size scale. In such case, the f-BIOPSY chip can beupgraded with an additional spiral channels (Triple Spiral shown in FIG.12B) for separating RBCs, leukocytes and CTCs using spiral microfluidicchannels 1211, 1212, and 1213, respectively. It is noted that althoughone or more interconnect channels are not explicitly shown in FIG. 12B,it should understood that spiral microfluidic channels 1211, 1212, and1213 may be respectively connected through interconnect channels.

The CTCs of larger sizes can be collected and used for experimentinggene expression profiling and further cell culture. The CTCs of smallersizes can be collected along with the leukocytes and can be sorted outusing immunomagnetic assays for genomic PCR or immunoprotein analysis.Although sample dilution is required in the f-BIOPSY system, the chipruns at high flow rate and the chip allows for easy parallelization withthe ability to analyze millilitres of clinical blood samples withinminutes.

A generalized the size based sorting technique in to a digitalcentrifuge by incorporating parallel multiple spiral based microfluidicsorting channels 1301, 1303, 1304, 1305 and 1306, using a single inlet1302 similar to traditional centrifuge system with the additionalprovision of low cost, automatic and robust platform is shown in FIG.13. It is noted that although one or more interconnect channels are notexplicitly shown in FIG. 13, it should understood that channels 1301,1303, 1304, 1305 and 1306 may be respectively connected throughinterconnect channels.

Serum Based Mobile Driven Analyzer for Rapid Tests

In the project on ‘Serum based Mobile driven Analyzer for Rapid Tests(SMART)’ shown in FIG. 14A-E, a SMART chip and system for Smart PhoneDriven Blood-Based Diagnostics is developed. In this system, personaldiagnostics information from whole blood is derived using a microfluidicchip and transmitted to cloud computing network for access to relevantusers. A serum separation chip using spiral fluidics actuated byacoustic cavitation streaming can be developed for calorimetric assays.The SMART chip can be integrated with mechanical, electronic, opticalcomponents for carrying out POC diagnostics powered by cloud computing.Biochemical techniques can be carried out off the chip to develop andoptimize colorimetric bioassay. Fluidic, electronic, optical andbioassay experiments can be performed on the chip independently andcollaboratively in order to evaluate the chip and system.

A compact disposable SMART chip can be developed to sort serum fromblood and to perform colorimetric assay on nitrocellulose pads. Therapid serum separation from whole blood is accomplished using cascadedserum extraction regions in spiral microfluidics (FIG. 15A). Theseparated serum enters multiple sides of cellulose nitrate pads forcarrying out colorimetric assays for diagnostics. In order to actuatethe pumping along the channels an acoustic cavitation streaming pump canbe utilized (FIG. 15B). This provides not only rapid assay but alsoenables low power operations. The serum extraction is carried out fromwhole blood inlet 1603 to serum outlet 1605 and cells outlet 1604 usingpassive microfluidics with faster protocol and undiluted blood using thetechnique shown in FIG. 16. The optimized geometry of the device withinterconnect channel 1602 can provide efficient separation with outclogging or sample dilution and extraction of plasma from whole humanblood. In this chip, the cell-free layer is considerably enhancedlocally by geometric singularities such as an abrupt enlargement of thechannel 1601 or a cavity along the channel. The extraction yield, theextraction purity, the flow rate stability regime and the range ofsample dilutions are optimized. In straight microchannels, neutrallybuoyant particles experience lateral migration in a circular Poiseuilleflow, resulting from two competing inertial Effects: one due to theinteraction with the wall, which produces a lift force away from thewall, and the other due to the shear and curvature of the Poiseuillevelocity profile, which induces migration. A magnified image of aportion of the SMART chip 1609 is shown. In the SMART chip multipleunits of extraction channel 1607 are arranged in a spiral fluidics forrapid and efficient extraction of serum. Whole blood entering primaryspiral microfluidic channel 1610, pinching region 1608 and serum isextracted through the “L” shaped interconnect channel 1606 to secondaryspiral microfluidic channel 1611.

FIG. 17 shows an alternative schematic for serum separation at output1704 from a whole blood from inlet 1701 using successive cell filtering.In this chip, several layers of spiral microfluidic channels withdecreasing width interconnect channels are shown. As illustrated in FIG.17, a spiral microfluidic system includes inlet 1701, primary spiralmicrofluidic channel 1710 and a set of secondary spiral microfluidicchannels (e.g., spiral microfluidic channels 1711, 1712, 1713, 1714 and1716), one or more groups of interconnect channels (e.g., interconnectchannels 1706, 1707, 1708 and 1702). The size of the interconnectchannels can filter smaller and smaller particles as the blood flowsinto adjacent spiral microfluidic channels. For example, the width of afirst group of interconnect channels 1706 connecting primary spiralmicrofluidic channel 1710 and secondary spiral microfluidic channel 1711is 10 um. The width of a second group of interconnect channels 1707connecting secondary spiral microfluidic channel 1711 and third spiralmicrofluidic channel 1712 is 5 um. The width of a third group ofinterconnect channels 1708 connecting third spiral microfluidic channel1712 and fourth spiral microfluidic channel 1713 is 3 um. The width of afourth group of interconnect channels 1702 connecting fourth spiralmicrofluidic channel 1713 and fifth spiral microfluidic channel 1714 is1.2 um. These spiral microfluidic channels with fractional turns can beseen as several primary or secondary spiral microfluidic channels withends having no outlet (e.g., ends 1703 and 1709) except outlet 1704 ofsixth spiral microfluidic channel 1716 connected to fifth spiralmicrofluidic channel 1714 through fifth group of interconnect channels1715. Serum is extracted in the last spiral and collected at the outlet1704.

The SMART system (described in FIG. 18) includes a microfluidic compactchip for serum separation from blood and colorimetric assay, a lenslessimaging for the quantification of personal diagnostics and a smartphone. The role of smart phone is two fold: 1. to provide electricalpower to fluidic, electronic and optical components of the SMART system2. to provide software control for acquisition, computation,communication and display of diagnostics data for the SMART system. Thedisposable compact chip performs the colorimetric assay for diagnosingblood. A lensless contact imaging approach is employed to obtain acompact imaging of the colorimetric assay with adequate resolution. Theimaging system composed of a housing for reaction chip positioning andlight shielding, which can dock a transparent microfluidics-basedreaction chip to image through a fiber optic taper. A chip holder withcover assured reproducible positioning of the chip during themeasurement and provides shielding from ambient light. Enzyme activitiescan be measured on cellulose pads inside the microfluidic chip. Thecamera can be controlled by the smart phone, by means of which lightemission intensity and 2D distribution data can be easily acquired andprocessed. As a proof of concept, the suitability of the device fordiagnostics can be demonstrated by performing models of the most commonclinical chemistry enzyme activity glucose assay.

The smart phone application software as described in FIG. 19, is acentral software control of the SMART system. The application softwareinterfaces to the hardware and provide communications to cloud networkand user for the display of diagnostic information. A Samsung GalaxyAndroid phone and communicate to Amazon EC2 cloud computing using JSONcommunication protocol is used. If the computational power is notsufficient for performing several image processing algorithms at smartphone, the heavy computation is performed at the cloud server. Otherwisesmart phone's computing power is used to process the images. The smartphone application software communicates with the Java middleware in thecloud and perform several image processing and computation to extractthe diagnostics data. Further the smart phone application softwarecollects the diagnostics data and display at the smart phone formonitoring or to alert the user or patient. GUI or user authorizationsoftware at the smart phone end as well as cloud portal is developed.The patients' health information is made available on the cloud networkfor physicians or health care provides.

Parallel Incubators with Loaded Single Cells for Lysis and AmplificationReactions

The single cells or molecules encapsulated picoreactors chip/system asshown in FIG. 20A-E for gene amplified enumeration and/or followed byelectrophoresis is critical to the development of pathology,oncogenesis, and other processes of a desired target cell or molecules.The system combines single cell trapping in micropillars, immisciblemicrofluidics, and fluorescent reverse transcription (RT)-PCRtechnologies performing molecular diagnostics in a quick,high-throughput, and cost-effective fashion. This highly integratedplatform is configured to:

-   -   precisely trap a single cell in array of configured micropillars    -   encapsulation of single cells as picoliter reactors using        immiscible microfluidics    -   simultaneously perform thousands of single-cell PCR in picoliter        volumes    -   rapidly analyze the PCR results on a chip    -   analyze single-cell of large populations for clinical        diagnostics or biomedical research        The combination of high-fidelity manipulation of single cells        and the ability to perform nucleic acid amplification offers the        possibility of developing powerful automated instruments.

The trapping site configuration with another set of shorter cylindricalmicropillars as guide posts (shown in FIG. 21) in front of the trappingsite locations by understanding the flow dynamics of the cells ismodified. Therefore efficiency of single cell trapping and reactorencapsulation can be much improved to trap single cells for a highthroughput of 10 k-100 k range. During encapsulation process, theseshorter guiding posts can be immersed in to the oil phase.

FIG. 22 shows the fluidics diagram of the massively parallel picoreactorchip with inlet splitter channels and outer merger channels forhomogeneous cells flow in each compartment. The cells and reagents areflowed from the top inlet. Extra cells or cell clusters adhered weaklynear the trap sites are cleaned by the flow at the side inlet. Using CFDsimulations, the picoliter reactor forming around trapping sites usingimmiscible fluids as shown in FIG. 23A-E, is proved. The geometry anddimension of the channel, the flow parameters for aqueous and oil flow,and electrical parameters for the droplet sorting and fusion areoptimized and preliminary results of single cell trapping andencapsulation of aqueous fluid using immiscible fluid is shown in FIG.24A-B

Single Cell Trapping Sites:

There are a few mechanisms of trapping sites for single cells. In onecase as in shown in FIG. 25, each single cell trapping site isconfigured by six square micropillars of dimension 5 um×5 um is arrangedso that a 10-15 um cell enter and get trapped while the fluid flowsaway. Additional micropillars are added to increase the volume of PCRmix solution in each reactor site. The 6^(th) micropillar at the bottomis added to increase the volume of the encapsulated PCR solution as wellas for the smooth formation of droplet. The pitch of the trapping sitesare optimized with different pitch in the x and y directions. Lesser thedistances the yield of trapping of the cells is better. If the distanceis too small there is choking of cells in the channel. If the distancesare more the cells freely flow through the device to the outlet andtrapping is limited. FIG. 26 shows the fluidic manifold for holding thePILLAR chip and flow based thermal cycling using a set of valves andpumps.

In another method each single cell trapping site is configured by a 5 umnozzle in the microchannel and many such trapping sites are connected inseries and parallel throughout the channel as shown in FIG. 27A-E. Thesingle cell trapping sites offer blockage to the flow and successivecells in the direction of the trapped cell. The cells are trapped asthey pass through the lower flow resistance or shorter channel. Thetrapped cells are valved so that successive cells flow through thelonger channel as shown in FIG. 28A-D. The number of the trapping sitesin series and parallel are optimized with the calculation of thepressure due to the flow of oil and the pump used. The sample withdisaggregated cells is loaded into the cell inlet in the microfluidicchip and is split in to many channels using binary splitters. The cellsare equally split in all the splitter channels. The cells from splitterchannels enter in to the trapping sites and finally merge in binarymerger channels to the outlet. The number of cells exiting the outlet isthe difference of the cells at the inlet and the number of cellstrapped. In a 1″×3″ area, ˜10000 sites can be accommodated along withother channels for fluidic delivery. FIG. 31A-C shows a single trappingsite (a), possible electrophoresis after PCR experiments (b), andpicoreactor droplet is coupled with EWOD to move the single cellencapsulated droplet for further serial processing (c).

Splitter/Merger Channels to Compartments:

The sample with disaggregated cells and PCR solution is loaded into thecell inlet in the microfluidic chip and is split in to many channelsusing binary splitters. The cells are hydrodynamically flowed along withPCR master mix and primers. The cells are equally split in all thesplitter channels. The cells from splitter channels enter in to thetrapping site compartments in a parallel fashion. Flow of fluid andcells enter in to serial compartments and finally merge in binary mergerchannels to the outlet. The number of cells entering the outlet is thedifference of the cells at the inlet and the number of cells trapped inmicropillar sites.

Compartmentalizing the trapping sites improves the homogeneity of theflow of cells within the trapping sites so that the yield of single celltrapping is improved. Placing the compartments serially in a serpentinechannel increases the fluidic resistance of the channel. So the flow ofcells in the chip is divided in to many subchannels using binarysplitters and the compartments are arranged in a parallel fashion. Themaximum number of trapping site in a compartment is limited by theparabolic fluidic flow. Using CFD simulation a maximum of 4 trappingsites are configured in the direction perpendicular to the flow. It isalso necessary to add sufficient margin in the compartment in all sidesfor placing the trapping sites. The number of trapping sites on thedirection of the flow is limited by the cell density. In a 10 mm×10 mmarea, ˜10000 sites can be accommodated.

Slip and Lock Chip:

The PCR reagents is be flowed through the top plate and is placed ontothe bottom plate in a air tight locked position secured using a Z-stageas shown in FIG. 29A-B or using electromagnetic based locking system.The ducts in the bottom plate were overlapped with the empty wells inthe top plate, forming a continuous fluidic path for loading of thesample. The top plate was then moved using a X-stage relative to thebottom plate to align the PCR reagent containing wells in the top platewith the single cell reservoirs in the bottom plate. Once the Slip andLock Chip is aligned, the PCR reagents and the single cells in bothplates are mixed by diffusion. After the mixing, the chip is locked inplace using the Z-stage and thermal cycling for PCR is started.

Chip Operation:

The chip is configured to perform five serial steps (1) Microarrayspotting of multiple primer pairs, (2) Flow based single-cell trappingusing micropillars, (3), Flow of immiscible fluid for forming picoliterreactors (4) convection driven thermal cycling for PCR, and (5)Fluorescence imaging based quantification and deletion analysis. Thesample of disaggregated cells along with PCR master mix and PCR solutionis loaded into the ‘cells inlet’ in the disposable microfluidic chipwhich sits on to the ‘fluidic manifold’. The manifold accommodates twooil baths, valves and a pump for oil delivery in to the disposable chip.The sample cells flow is split in to many channels and each single cellis trapped at the micropillars trapping sites. The trapped single cellsis encapsulated by flowing oil at RT from cold bath and the oil flowfront goes around the micropillars as shown in the CFD simulation. Theencapsulated reactor includes single cell, spotted PCR primers andencapsulated PCR master mix with lysing buffer and enzymes. In order toperform PCR thermal cycling oil from isolated hot and cold baths isheated at temperatures 95° C. and 50° C. and is circulated into theentrapped droplets alternately using the pump. Using CFD simulations itis understood that the sealed picoreactors locked at the micropillarsites is immobile with temperature or pressure effects. A heating rateof 1° C./ms and a cooling rate of 2° C./ms are expected during thethermocycling with the circulation of oil. The fluorescence detectionsystem includes a tungsten-halogen/mercury lamp as an excitation sourceand a CCD detector with an XY stage. After loading the cells andreagents, the chip is operated automatically using Labview softwarethrough NI-DAQ interface for final fluorescence analysis of themultiplexed PCR.

System Integration: The system includes a flow device, fluorescencedetection, thermal cycler, and software control systems. The flow deviceis facilitated with Pico syringe pumps (Harvard Apparatus, MA) fordelivering fluids into the channel with a constant flow rate between 10μl/min and 100 nl/min. The PCR is performed using an externally appliedprogrammable Peltier heater. A Peltier heating element (Melcor) controlsthe nanodroplet temperature between 30 and 95° C. A heating rate of 6-8°C./s and a cooling rate of 2-4° C./s are expected. The timing of thethermal cycling (94° C., 60° C., and 72° C.) is also controlled by thesoftware. The fluorescence detection system includes a tungsten-halogenlamp as an excitation source and a CCD detector (Spectral InstrumentsInc., Tucson, Ariz.). Various optical filters are used to accommodatefor different fluorescence dyes. The common dyes are FAM, VIC, TAMRA,SYBR Green, JOE, etc. The wavelengths of excitation light are 470 nm,490 nm, 530 nm, and 635 nm. Since the fluorescence signal is amplified,the CCD has sufficient sensitivity for fluorescence detection. If thearray area is too big (>1 cm²) for single illumination, a scanningmechanism is facilitated for multiple illuminations.

All the components are programmed using Labview software through NI-DAQinterface. After loading the cells and reagents, the chip is operatedautomatically for single-cell encapsulation, generating PCR samples,performing temperature control for PCR cycles and final fluorescenceanalysis of the PCR data. Diagnostics and prognostics analysis throughcopy number variation are performed using multiple single cell PCRresults. A diagram showing the diagnosis of diseases using fluorescencesignal with highlighted positive samples is shown in FIG. 30.

Post-Processing of Docked Picoreactor:

Using ‘electrowetting on dielectric’ (EWOD), the docked picoreactors intrapping sites with single cell encapsulation can be moved for furtherserial or multistep processing. In order to accomplish the movement ofsuch droplets, EWOD electrodes assembly is laid on the bottom layer ofthe chip. Further by replacing the oil by a gel medium electrophoresis(with electrodes on glass plate) can be perform after the PCR reactionas shown in FIG. 31C.

Programmable Array of Living Cells

The purpose of this proposal is to develop and commercialize amicrofluidic Programmable Array of Living Cells (PAL) for CombinatorialDrug Screening. The overall goal in PAL innovative technology is todemonstrate a low cost, high throughput, multiplexed, automated,integrated, passive, scalable, portable system for cancer therapeutics.The PAL chip can be fabricated for combinatorial fluidics for 16×16 cellbased assay reactors. This chip can enable us to perform 2 drugs at 16concentration or 4 drugs at 8 concentrations for combinatorial drugscreening. In this PAL system, ˜100 cells are captured by cup shapedpillars and are encapsulated by immiscible fluids as virtual wall inorder to isolated different reactors for cell growth monitoring underthe influence of drug cocktails. The PAL system (as shown in FIG. 32A-K)is configured to perform four serial steps (I) Flow of cells to fill the‘cup’ shaped reactor (II) Flow of buffer for cells cleanup and flow ofcombinatorial drug. (III) Flow of oil to form cells encapsulatedreactors (IV) Cell culture with florescence imaging based cell growthmonitoring.

In the single layer pillars chip, the immiscible fluid is very close tothe cells. The immiscible fluid, fluorinert is biologically inert andmay not interfere with the cell membrane. However, an alternategeometry, as in FIG. 32L, with double layer pillars with cells foundonly in the inner layer in the 24-72 hours of experiment is presented.The inner layer pillars can be fabricated with smaller (˜5 um) spacingwhile the outer pillars can be fabricated with larger (10-20 um) so thatthe cells are captured only in the inner compartment. The outer layerpillars can encapsulate sufficient cell media/nutrition for continuousculture for several days. Another feature is an overflow “cup”arrangement for the cells at the top of the inner layer pillars. The gapbetween the funnel and the cup can be optimized to avoid any clogging ofcells. This system can ensure that equal amount of cells can be capturedin each reactor.

EXAMPLE 1 IPSC Sorting for Stem Cell Therapy

The derivation of patient-specific reprogrammed somatic cells makesimmunologically compatible stem cell replacement strategy veryattractive for several applications such as spinal cord therapy. Inpotential therapeutic applications, the cell populations relevant fortherapy can be electrically excited and the resulting transmembrane ioncurrents are measured using an array of surface microelectrodes alongthe direction of the flow. The electrical current measurements inresponse to electrical stimulation for differentiated states of thecells are built up as electrical signatures for real time comparison andsorting. Since these transmembrane ion currents are measurednon-invasively to sort the differentiated cells based on these fieldpotential markers, the sorted cells are highly viable for therapeuticapplications. The iPSC line to be studied can be derived from fibroblastsample SC-140 cells. These cells are maintained in StemPro medium onMatrigel-coated plates. The cells are diluted to make a concentration of2,000 cells per L before using the OFFSET system. These cells aredifferentiated into neural stem cells, neurons, and glia and after thedifferentiation, the cells are maintained in appropriate media. Thecells are non-enzymatically removed from the plates and immediatelyintroduced into the microfluidic device for sorting. All of the typicalcriteria needed for clinical cell production can be incorporated intosuch a process, including donor screening, raw materials sourcing,vendor qualification, process documentation, and assay and processqualification. In this approach, each step of the process must meet itsown criteria such that the trajectory from starting material to finalproduct is sufficiently well-characterized that a full characterizationof the final product becomes unnecessary.

EXAMPLE 2 Diagnostics of Cancer

The analysis of heterogeneity in individual tumor cell represents amajor step in developing a precise molecular signature of a patient'scancer which leads to therapies tailored to individual patients, animportant objective for new oncology drugs. At such single cell level,preamplification of the entire mRNA library to analyze a multigenereverse transcription-PCR panel without compromising the sensitivitiesof individual marker genes is required. Circulating tumor cells (CTCs)that circulate in the bloodstream alongside normal cells represent a“real-time” biopsy with a surrogate source of tissue in cancer diagnosisand prognosis. The inhomogeneities in the tumor cells and their flow into blood stream require interrogation of the individual tumor cells andcomparison of individual CTCs' expression levels. The ability toquantify and profile the gene expression of CTCs allows improvedbiological characterization of cancer diagnostics in real time andexpedite the development of effective patient-specific therapies.Automated enumeration and characterization of multigenes in circulatingtumor cell (CTC) from whole blood have widespread implications in theprognosis and diagnosis of cancer. Furthermore, with the ability tomultiplex in a massively parallel fashion, several genes can be screenedsimultaneously and each panel can also contain desired positive andnegative controls. Assays to detect cancer cells in blood have been usedclinically to provide prognostic and theranostic information and to testfor minimal residual disease. This system has the capacity to processthousand of individual cells; detect circulating tumor cells based onmultiplexed PCR results; and analyze the presence or absence of themultiple genes. The presence of circulating tumor cells in the blood canbe detected at the single cell level in a population with thousands ormore cells by applying single cell PCR assays using expressed mRNA ormicro RNA and Philadelphia chromosome positive acute lymphoblasticleukemia (Ph+ALL) are characterized at the molecular level by thebcr-abl fusion transcript. The early detection of mutations in theBcr-Abl fusion tyrosine kinase by SC-PCR in a cell population may allowtimely treatment intervention to prevent or overcome resistance.

EXAMPLE 3 Diagnostics Using Clinical Blood Samples

Diagnostics using clinical samples is important for routine andnon-invasive testing of patients undergoing therapy. For example,Hepatitis C virus (HCV), is a major cause of chronic liver disease, withan estimated 200 million people affected worldwide. Despite recentsuccess after the introduction of combination therapy with interferon(IFN)-α and ribavirin, resistance to antiviral therapy remains a seriousproblem in the management of chronic hepatitis C. The absence of HCV inthe serum of patients by the end of treatment, does not exclude futureviremia. The most extrahepatic site for the virus is peripheral bloodmononuclear cells (PBMC) and these cells are considered as a potentialreservoir of HCV infection. The patient might still be a source ofinfection to others and so it is strongly encouraged to test for HCV inPBMC to detect lack of response to treatment and persisting infection.Ultrasensitive and specific non-invasive and risk-free monitoringsystems that measure very low levels of HCV in blood have been ofgreater significance for diagnostics and prognostics. The programmablemicroarray based single cell diagnostics system performs rapid,sensitive, specific and reproducible quantitative monitoring of HCV RNAin PBMCs. This new technology featured by processing very minute amountof samples and reagents has the potential to detect wide variety ofliver diseases simultaneously in the frequency domain by digitallyanalyzing statistically significant samples. The system is useful notonly for the diagnostics but also for therapeutics and discovery of newvaccine which has also been hampered by the great heterogeneity of theHCV genome.

EXAMPLE 4 Diagnosis of Infectious Diseases

Diagnostics of infectious diseases requires and automatic one touchanalysis of blood sample or other cells. Precise molecular analysis onsingle cells from a large population of cells led to the enumeration ofcells with specific genes. For example HIV/AIDS diagnosis can beperformed by analysis of single PBMC cells for the presence or absenceof cell-associated HIV viral genomic RNA and the mRNA of β-actin.Researchers often purify DNA from blood samples prior to performing PCRbecause it is believed that blood constituents and the reagents commonlyused to preserve blood samples (e.g., anticoagulants) interfere withPCR. But in the case of single cell PCR such purification process is notrequired and high throughput such PCR reactions can be used for theenumeration of CD4 cells which are specifically targeted and destroyedby HIV. A healthy person's CD4 count can vary from 500 to more than1,000. Even if a person has no symptoms, HIV infection progresses toAIDS when his or her CD4 count becomes less than 200. Prompt diagnosisand treatment can reduce or delay the onset of some seriouscomplications, such as opportunistic infections, and can improve qualityof life. In some cases, rapid treatment with medication can prevent thedevelopment of HIV/AIDS after exposure to the HIV virus. Normal PBMC andhuman immunodeficiency virus (HIV) type-1 infected PBMC cells can bedistinguished in RT-PCR. The mRNAs released from the cell are reversetranscripted into cDNA using Sensiscript™ Reverse Transcriptase in theenzyme mix.

EXAMPLE 5 Diagnostics of Prenatal Diseases

The system is used for various genetic diseases or syndrome at prenataldiagnosis. For example, muscular dystrophy refers to a group of morethan 30 genetic diseases that involve mutations in any of the thousandsof genes that program proteins critical to muscle integrity resultingdegeneration of skeletal muscles towards death. Duchenne Musculardystrophy (DMD), caused primarily by intragenic deletion or duplicationshas no treatment as of now and prenatal diagnosis is the most importantpreventive strategy. DMD alone affect approximately 1 in every 3,500 to5,000 boys or between 400 and 600 live male births each year in theUnited States. Detection of a DMD gene mutation is sufficient toestablish a diagnosis of DMD and so multiplex PCR method is the bestdiagnostic tool owing to its characteristics such as specific, accurate,sensitive and rapid. Presently, the prenatal diagnosis of DMD isperformed through deletion analysis using DNA extracted followingamniocentesis or chorionic villous sampling (CVS). After sampling, CVSare microscopically dissected and after homogenization, DNA is extractedand controlled with multiple polymorphic markers to ensure its fetalorigin and to avoid maternal tissue contamination, which could possiblyresult in inaccurate results. The massively parallel microspatiallyaddressed multiplexed PCR system performs fast frequency domain sampleanalysis of DMD from prenatal samples at high reliability, accuracy andspecificity to validate the clinical efficacy and practical feasibilityamong high risk pregnancies. The prenatal sample may contain maternaltissue contamination which are eliminated by analyzing multiple singlecell PCR analysis. Further, multiple polymorphic markers are employed toensure its fetal origin in multiplex PCR to analyze prenatal DMDdiagnostics. This distinguishes between maternal tissue contaminationand CVS cells and confirms the single cell PCR performance for deletionsanalysis of true CVS cells.

Single-cell multiplex PCR is performed using HotStarTaq™ DNA Polymerase(Qiagen, Valencia, Calif.) following the guidelines for single-cell PCRgiven in the HotStarTaq PCR Handbook (Qiagen). Fluorescent multiplexsingle cell PCR protocol for different mutations of DMD gene isanalyzed. Single cells are analyzed for the presence or absence of theexons 45, 48, 49, 43, 19, 3, 8, 13 and the promoter region of the humandystrophin gene for comparison. The cells loaded in the chip along withlysis buffer and PCR master mix react with the dried primers spots inthe chip during the PCR amplification. The cDNA are amplified withfluorescent PCR, and fluorescent signals are detected by the fluorescentscanner.

EXAMPLE 6 Applications in Biomedical Research

Single-cell PCR has proven to be of enormous use to basic scientists,addressing diverse immunological, neurological, and developmentalquestions, where both the genome and also messenger RNA expressionpatterns are examined. Enhancements in sensitivity with SC-PCR permitsscientists to investigate changes at the level of a single cell, farbelow what are needed using traditional methods. The understanding ofmany biological processes would greatly benefit from the ability toanalyze the content of single cells.

The system can be used to screen the gene expression variation of fewsignificant genes for regenerative medicine in large number of singlecells. The gene expression profile of Nanog and Oct-4 (positive genesfor the undifferentiated state) and Pax6 and Sox1 (positive genes forthe differentiated state) from SC-140 iPS cells is used qualiy iPSC.

The advantage of diagnosing a patient's cancer at the single cell levelprovides us an approach for early detection of cancer and yield insightsinto how cancer cells are responding or adapting to therapy. An extendedsingle cell technique predicts the pathways of cancer cells thatcircumvent current therapies and direct the patient towards alternativetreatments more intelligently.

The goal in forensic science is to eliminate uncertainty, usingtechnology to precisely determine identity. Researchers continue torefine and improve forensic methods using single cell analysis withsuccess for both increased sensitivity and cost savings.

Fetal cells can be found circulating in maternal blood. Fetal cellsrecovered from maternal blood provide the only source for noninvasiveprenatal DNA diagnosis. Recently, genetic diagnosis using fluorescentPCR has been applied at the single-cell level for sex or single-genedefect diagnosis

Circulating tumor cell levels in blood may serve as a prognostic markerand for the early assessment of therapeutic response in patients withmetastatic cancer, and are an independent prognostic factor at primarydiagnosis. The presence of circulating tumor cells in the blood can bedetected at the single-cell level, by applying single-cell PCR assays.

This technology can be extended to diagnostics of various diseases.Small concentration changes and/or altered modification patterns ofdisease-relevant components, such as mRNA and/or micro RNA, have thepotential to serve as indications of the onset, stage, and response totherapy of several diseases. Current single cell PCR methods useindividual cells of interest isolated by micromanipulation or cellsorting. Low abundance mRNA is often lost during cell lysis andextraction process. These methods are extremely labor intensive andrequire expensive equipment to isolate single cells and perform PCR oneach cell. However, to detect rare abnormal cells, a large number ofcells must be analyzed spontaneously. There is “no” current method thatcan process, characterize, and perform qRT-PCR for thousands of singlecells simultaneously and quickly in picoliter volume.

EXAMPLE 7 Smartphone Based Applications

Cancer Diagnostics: Smart phone based diagnostics would help in thedetection of drug adverse reaction during cancer therapy. Currently, 60%of patients diagnosed with breast, colon, lung, or ovarian canceralready have cell metastases forming in other locations of their body.In addition to antibodies for cancer cell detection, other mechanismssuch as peptides and aptamers are used.

HIV Detection: Smart phone based HIV diagnostics can help in the earlydetection of HIV in remote villages in such countries by health workers.More than 30 million HIV-infected people live in the developing world,where resources are scarce. To increase access to HIV care and improvetreatment outcomes, there is an urgent need for low cost diagnostictools that could be implemented in developing countries. The device canalso be engineered to detect other virulent pathogens, includinghepatitis B and H1N1 (swine) flu.

Clinical Diagnostics: Smartphone based clinical diagnostics can be usedfor archiving and monitoring diagnostics data for several diseases so asto provide health information to patients, physicians and healthcareproviders. Several clinical diagnostics panels can be used for routinetelemedicine. Detection of C-reactive proteins (CRP) in the blood, apreferred method for helping doctors assess the risk of cardiovascularand peripheral vascular diseases can help in saving the life of manyseniors and other patients through smart phone based telemedicine. Thenumber of CRP tests paid for by Medicare tripled from 145,000 to 454,000(from 2002 to 2004), and it is estimated that those numbers havequadrupled since then.

It is to be appreciated that the present invention has been describedhereabove with references to certain examples or embodiments of thepresent invention but that various additions, deletions, alterations andmodifications may be made to those examples and embodiments withoutdeparting from the intended spirit and scope of the present invention.For example any element or attribute of one embodiment or example may beincorporated in to or used with another embodiment of example unsuitablefor intended use. All reasonable additions, deletions, modification andalterations are to be considered equivalents of the described examplesand embodiments and are to be included within the scope of the followingclaims.

What is claimed is:
 1. A spiral microfluidic channel system comprising:an inlet for flowing a plurality of cells; a primary spiral microfluidicchannel coupled to said inlet, said primary spiral microfluidic channelcomprising a plurality of pinching regions; one or more outlets forseparating said plurality of cells.
 2. The spiral microfluidic channelsystem of claim 1, further comprising one or more secondary spiralmicrofluidic channels coupled to said primary spiral microfluidicchannel.
 3. The spiral microfluidic channel system of claim 2, whereinsaid primary spiral microfluidic channel is coupled to at least one ofsaid one or more secondary spiral microfluidic channels through aplurality of interconnect channels.
 4. The spiral microfluidic channelsystem of claim 3, wherein said plurality of interconnect channelsconnect said at least one of said one or more secondary spiralmicrofluidic channels to said plurality of pinching regions of saidprimary spiral microfluidic channel.
 5. The spiral microfluidic channelsystem of claim 2, wherein said primary spiral microfluidic channel andsaid one or more secondary spiral microfluidic channels aresubstantially concentric.
 6. The spiral microfluidic channel system ofclaim 1, wherein said primary spiral microfluidic channel has adecreasing width from said inlet to at least one of said one or moreoutlets.
 7. The spiral microfluidic channel system of claim 2, whereinat least one of said one or more secondary spiral microfluidic channelshas an increasing width from said inlet to at least one of said one ormore outlets.
 8. The spiral microfluidic channel system of claim 1,wherein said plurality of pinching regions are periodically disposedalong a convex or concave sidewall of said primary spiral microfluidicchannel.
 9. The spiral microfluidic channel system of claim 2, whereinsaid primary spiral microfluidic channel is coupled to one of said oneor more secondary spiral microfluidic channels through a first pluralityof interconnect channels.
 10. The spiral microfluidic channel system ofclaim 9, wherein said one of said one or more secondary spiralmicrofluidic channels is coupled to another one of said one or moresecondary spiral microfluidic channels through a second plurality ofinterconnect channels smaller than said first plurality of interconnectchannels, such that larger cells from said plurality of cells aretrapped in said primary channel and smaller cells from said plurality ofcells are passed to said one or more secondary channels.
 11. The spiralmicrofluidic channel system of claim 1, wherein said primary spiralmicrofluidic channel is configured to separate circulating tumor cells(CTCs) from said plurality of cells.
 12. The spiral microfluidic channelsystem of claim 2, wherein said at least one of said one or moresecondary spiral microfluidic channels is configured to separate redblood cells (RBCs) from said plurality of cells.
 13. The spiralmicrofluidic channel system of claim 3, wherein at least one of saidplurality of interconnect channels is an L-shape interconnect channel.14. The spiral microfluidic channel system of claim 13, wherein saidL-shape interconnect channel connects one end of at least one of saidplurality of pinching regions to at least one of said one or moresecondary spiral microfluidic channels.
 15. A method comprising:providing a plurality of cells to an inlet; sorting a first group ofcells from said plurality of cells using a first spiral microfluidicchannel coupled to said inlet; sorting a second group of cells from saidplurality of cells using a second spiral microfluidic channel; whereinsaid second spiral microfluidic channel is coupled to said first spiralmicrofluidic channel through a plurality of interconnect channels. 16.The method of claim 15, wherein said sorting said first group of cellsand said sorting said second group of cells from said plurality of cellsare based on inertial microfluidics.
 17. The method of claim 15, whereinsaid plurality of interconnect channels connect said second spiralmicrofluidic channel to a plurality of pinching regions of said firstspiral microfluidic channel.
 18. The method of claim 15, wherein saidfirst spiral microfluidic channel and said second spiral microfluidicchannel are substantially concentric.
 19. The method of claim 15,wherein said first spiral microfluidic channel has a decreasing widthfrom said inlet to an outlet.
 20. The method of claim 15, wherein saidsecond spiral microfluidic channel has an increasing width from saidinlet to an outlet.